Summary of work: The insulin receptor is a member of the protein tyrosine kinase family that plays an important role in diverse physiological processes, including modulation of glucose homeostasis and gene expression. Earlier findings have found that the functional activity of the insulin receptor may be modulated by redox regulation in the cellular environment. An increase in plasma free radical production combined with a significant reduction in plasma glutathione (GSH) levels have been associated with a reduction in insulin action both in the elderly and type 2 diabetic patients. It is believed that alteration in glutathione redox status is likely to be an important factor which may account for the pathologic insulin resistance, a common feature of type 2 diabetes and aging. Indeed, administation of GSH, which constitutes the major source of plasma nonprotein thiols, significantly improves the modulation of glucose homeostasis by insulin by raising the plasma GSH/GSSG ratio in diabetics. Because it does not penetrate cells readily, we hypothesized that GSH may play a role in structural and/or functional changes in the insulin receptor, thereby altering insulin responsiveness. In this study, we show that treatment of cultured cells with GSH but not the oxidized GSSG derivative reduces insulin receptor alpha-subunit thiols in a dose- and time-dependent manner. Removal of GSH from the incubation medium rapidly restores the native thiol redox status of the insulin receptor alpha subunit. N-acetylcysteine (NAC), a widely used modulator of thiol levels, acts as a cytoprotective drug with multiple therapeutic applications, in part, by acting directly as a reducing agent. Like GSH, NAC treatment resulted in an increase in the level of thiol reactivity in the insulin receptor alpha subunit that was reversed following NAC removal. The oligomeric structure of the insulin receptor was not affected by GSH or NAC treatment. Of interest, the enhancement in reactivity of the insulin receptor alpha-subunit thiols with GSH or NAC had little impact on insulin binding but selectively increased insulin-stimulated change in insulin signal transduction. Progress is being made in the identification of the reactive receptor thiol group(s). We plan to assess thiol reactivity of insulin receptors isolated from type 2 diabetics and aged animals before and after GSH or NAC administration.